Breeding habitats, bionomics and phylogenetic analysis of Aedes aegypti and first detection of Culiseta longiareolata, and Ae. hirsutus in Somali Region, eastern Ethiopia

Introduction Arboviral diseases, such as dengue, chikungunya, yellow fever, and Zika, are caused by viruses that are transmitted to humans through mosquito bites. However, the status of arbovirus vectors in eastern Ethiopia is unknown. The aim of this study was to investigate distribution, breeding habitat, bionomics and phylogenetic relationship of Aedes aegypti mosquito species in Somali Regional State, Eastern Ethiopia. Methods Entomological surveys were conducted in four sites including Jigjiga, Degehabur, Kebridehar and Godey in 2018 (October to December) to study the distribution of Ae. aegypti and with a follow-up collection in 2020 (July-December). In addition, an investigation into the seasonality and bionomics of Ae. aegypti was conducted in 2021 (January-April) in Kebridehar town. Adult mosquitoes were collected from indoor and outdoor locations using CDC light traps (LTs), pyrethrum spray collection (PSCs), and aspirators. Larvae and pupae were also collected from a total of 169 water-holding containers using a dipper between October and November 2020 (rainy season) in Kebridehar town. The species identification of wild caught and reared adults was conducted using a taxonomic key. In addition, species identification using mitochondrial and nuclear genes maximum likelihood-based phylogenetic analysis was performed. Results In the 2018 collection, Ae. aegypti was found in all study sites (Jigjiga, Degahabour, Kebridehar and Godey). In the 2020–2021 collection, a total of 470 (Female = 341, Male = 129) wild caught adult Ae. aegypti mosquitoes were collected, mostly during the rainy season with the highest frequency in November (n = 177) while the lowest abundance was in the dry season (n = 14) for both February and March. The majority of Ae. aegypt were caught using PSC (n = 365) followed by CDC LT (n = 102) and least were collected by aspirator from an animal shelter (n = 3). Aedes aegypti larval density was highest in tires (0.97 larvae per dip) followed by cemented cisterns (0.73 larvae per dip) and the Relative Breeding Index (RBI) was 0.87 and Container Index (CI) was 0.56. Genetic analysis of ITS2 and COI revealed one and 18 haplotypes, respectively and phylogenetic analysis confirmed species identification. The 2022 collection revealed no Ae. aegpti, but two previously uncharacterized species to that region. Phylogenetic analysis of these two species revealed their identities as Ae. hirsutus and Culiseta longiareolata. Conclusion Data from our study indicate that, Ae. aegypti is present both during the wet and dry seasons due to the availability of breeding habitats, including water containers like cemented cisterns, tires, barrels, and plastic containers. This study emphasizes the necessity of establishing a national entomological surveillance program for Aedes in Somali region.


Introduction
Arboviral diseases such as dengue, chikungunya, yellow fever, and Zika, are caused by viruses that are spread to people by the bite of an infected arthropod, mosquitoes.However, these diseases are considered neglected diseases and low priority has been given to their research in past decades.Due to the urbanization, globalization, and increased human mobility, unprecedented emergence of arboviral disease epidemics have been increased over time, and thus the need to carry out surveillance and research related to the arboviral diseases, and their vectors has risen [1].
Mosquito-borne viral infections such as yellow fever (YF), dengue fever (DF), and chikungunya (CHIK) are occurring frequently in some areas of Ethiopia.In recent years, outbreaks of DF occurred in the eastern, southeastern, southern and northwest parts of the country [2][3][4][5].Several factors are related to the increase of DF incidence in Ethiopia.Among the most important ones are uncontrolled urbanization and the absence of standardized public services, such as water supply, sewage and waste disposal [3].
Aedes aegypti and Ae.albopictus are the known vectors of dengue virus (DENV) and other arboviruses including chikungunya virus (CHIKV) and zika virus (ZV) [6].These species are highly efficient vectors of arboviruses and live in close proximity to humans [7].Ae. africanus and Ae.luteocephalus also act as potential vectors in Africa [8].Ae. aegypti is uniquely adapted to a close association with humans, which facilitates efficient virus transmission.Immature forms of the mosquitoes develop primarily in man-made containers in and around the homes of people [9,10].It is a highly anthropophilic, endophilic and endophagic day-biting species that rests inside houses where females feed frequently and preferentially on human blood [11].These behavioral traits enhance human-mosquito contacts and dengue virus attack rates to be very high [12].
In 2017 an outbreak of dengue fever was reported in Kebridehar Town in the Somali region of Ethiopia [13].In this study, a total of 101 cases occurred including five with severe dengue and one death were reported from 10 villages of the town.Despite the public health significance and the recent occurrences of dengue fever in this particular urban area, no detailed entomological studies to determine the breeding habitats, bionomics, and population genetic structure of Ae. aegypti were conducted.Therefore, the current study was designed to investigate the distribution, breeding habitats, bionomics, and genetic diversity of the of Aedes aegypti in Somali Region, Ethiopia.

Mosquito collection
Adult mosquitoes were collected from indoor and outdoor using Centers for Disease Control miniature light traps (John W. Hock, Gainesville, FL) (CDC LT), pyrethrum spray collection (PSC) techniques and mouth aspirator from animal shelters (Cattle and Goat) in four urban areas of Somali region in 2018, 2020 and 2021.In 2018 (October to December), adult mosquito collections were carried out in four sites including Jigjiga, Degahabur, Kebridehar and Godey (Fig 1).In addition, mosquitoes were also collected in Kebridehar from July-December of 2020 and January-April of 2021.Finally, a follow-up survey was conducted in Godey and Jigjiga 2022.Jigjiga University research review committee approved the study and a support letter was obtained from Somali Regional health bureau for mosquito house collection.Verbal informed consent was sought from participants prior to enrolling their house in the study.

CDC light trapping (CDC LT)
Twenty houses were selected for indoor and outdoor light trap catches from the same villages of Kebridehar town based on their relative location from the breeding site in a similar way to houses selected.Mosquitoes were collected from both indoor and outdoor locations from 6:00 pm to 6:00 am from each selected house using standard CDC LT.Traps were hung from the ceiling or from roof supports at the foot end of the bed where people sleep at night and the body of the trap was suspended about 1.5 meters from the floor.
Collection bags were attached to the trap by stretching the open end of the bag over the bottom rim of the trap and a label marked with the date and the site number was placed inside the collection bag.Collection bags were retrieved from traps in each house in the morning from 8:00 am to 9:00 am by entomological assistants.

Pyrethrum Spray Sheet collections (PSC)
Indoor resting Aedes sp. were sampled in the morning (6:00 to 8:00 Am) from 20 randomly selected houses in each study area by the application of pyrethrum spray catches.In PSC monthly mosquito collections were done in the same houses throughout the study period.Before spraying in each house, all food items were removed; the windows and door openings and eaves were filled with pieces of cloth, and the floor was covered with white cloth sheets.After the door was closed, the pyrethrum aerosol was sprayed for about three to five minutes.After ten minutes of spraying, the knockdown Aedes mosquitoes were then collected from the white sheets using fine forceps and placed in Petri dish for later processing and identification.Adult female Aedes mosquitoes were categorized into unfed, fresh fed, semi-gravid, and gravid according to their abdominal status under a dissecting microscope.

Mouth aspirator collection
Aedes mosquitoes were collected using mouth aspirator in the early evening and morning from an animal shelter, and other locations.

Processing of adult mosquitoes
Every morning during the collection period, mosquitoes captured in CDC LT, PSC, and aspirator from were transported to a field station laboratory and mosquitoes were transferred to test tubes.They were then knocked down using chloroform and emptied on petri dishes for sorting into distinct categories (genera, sex, and abdominal status) under a dissecting microscope.The adult mosquitoes were identified morphologically using a standard key [14].

Larval and pupae collection
A house-to-house mosquito breeding habitat survey was conducted in Kebridehar Town.Larvae and pupae were also collected from a total of 169 water-holding containers using a dipper between October and November 2020 in Kebridehar (rainy season) (Fig 2).Some other larvae and pupae mosquito specimens were collected in Jigjiga June 2022 that were morphologically similar to Aedes but not initially identified (S1 and S2 Figs).
Larvae and pupae of mosquitoes were collected from breeding habitats such as cemented cisterns, water containers, discarded tires, plastic containers and barrels.The presence of immature stages of mosquitoes was visually evaluated in all water-holding containers present in the premises of the houses.The number and type of containers inspected were recorded, including information on which are present or absent immature stages of mosquitoes.During sampling, 20 dips were made using a standard dipper from each positive breeding habitat.The collected immature stage was transported to a field laboratory.All collected larvae and pupae were reared in the laboratory until adults emerged, and all adults that emerged from the pupae were collected and stored in vials and carefully classified by species according to the pattern of white bands, using a dissecting microscope and identification keys [14].

Larval indices analysis
The larval survey data were calculated and analyzed in terms of different larval survey techniques like Container Index (CI), and Breteau Index (BI).The calculation of larval indices is based on the following mathematical formulae:

DNA extraction and molecular identification of mosquitoes
Molecular species confirmation was performed on a subset of specimens collected from Kebridehar, Degehabur, and Jigjiga.DNA was extracted from the abdomens selected at random, using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA).Polymerase chain reaction (PCR) was performed for each individual mosquito, targeting the nuclear internal transcribed spacer 2 (ITS2) region.The reagent components and concentrations for the PCR assays were 1X Promega HotStart Master Mix (Promega, Madison, WI) and 0.5 mM for both primers, plus 1 μL of isolated DNA template.Primers used in these ITS2 reactions were 5.8SB-5'ATG CTT AAA TTT AGG GGG TAG TC-3' and 28SB 5'-ATG CTT AAA TTT AGG GGG TAG TC-3'.The PCR protocol consisted of 95˚C for 2 min, 31 cycles of 95˚C for 1 min, 49˚C for 30 sec, and 72˚C for 1 min, followed by an extension step of 72˚C for 5 min.
The cytochrome c oxidase subunit 1 (COI) was amplified to determine any polymorphisms.Primers used in this reaction included LCO1490F 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198R 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.A negative control of no DNA template was used to ensure no contamination of PCR reagents.The reagent components and final concentrations for the PCR assays were 1X Promega HotStart Master Mix (Promega, Madison, WI) and 0.5 mM for both primers, plus 1 uL of isolated DNA template.
For COI, the PCR protocol was as follows: 95˚C for 1 min, 31 cycles of 95˚C for 30 sec, 48˚C for 30 sec, and 72˚C for 1 min, followed by an extension step of 72˚C for 10 min.Amplicons were sequenced using Sanger technology with ABI BigDye TM Terminator chemistry (Thermofisher, Santa Clara, CA) according to manufacturer recommendations and run on a 3170xl Genetic Analyzer (Thermo Fisher, Santa Clara, CA) through Eurofins Genomics (Louisville, USA).

Sequence and phylogenetic analysis
Sequences were cleaned and analyzed using CodonCode (CodonCode Corporation, Centerville, MA, USA).ITS2 and COI sequences were submitted as queries to the National Center for Biotechnology Information's (NCBI) Basic Local Alignment Search Tool (BLAST) against the nucleotide collection under standard parameters (100 max target sequences, expect threshold 0.05, word size 28, optimized for highly similar sequences, not specific to any organism).
Alignments were created with MAFFT version 7 (Katoh K, Rozewicki J, Yamada KD.MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization.Brief Bioinform.2019;20:1160-6.)using Codon Code version 8.01 (CodonCode Corporation, Dedham, MA, USA) and Mesquite version 3.61 (Wane P. Maddison DRM: Mesquite: a modular system for evolutionary analysis.2021.http://www.mesquiteproject.org.).Phylogenetic associations between sequences were estimated using a maximum likelihood approach with RAxML (Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models.Bioinformatics.2006; 22:2688-90).The GTRGAMMA option that utilizes the GTR model of nucleotide substitution with gamma model of rate of heterogeneity was used.One thousand runs were completed using the maximum likelihood criteria with rapid bootstrap analysis.The RAxML output was viewed in Fig- Tree, with a root at the out group, and a phylogenetic tree image was made.

Results
In total, 543 Ae. aegypti were captured from urban areas during various collection years (Table 1).During the 2018 collection, Ae. aegypti was recorded in all study sites and the highest number of Ae. aegypti was found in the town of Kebridehar (n = 35), followed by Godey

Larval density
In total 1419 larvae of Aedes mosquitoes and 329 pupae were sampled from cemented cisterns, tires, plastic containers, and barrels (Table 2).Aedes larval density was highest in tires (0.97 larvae per dip) followed by cemented cisterns (0.73 larvae per dip).The Relative Breeding Index (RBI) was 0.87 and Container Index (CI) was 0.56.Container locations were also observed during the entomological assessment, along with water status and types.The majority of the water holding containers that were positive for larvae/pupae were placed outside within an average 10-meter radius of the houses.

Phylogenetic analysis of specimens
For the morphologically identified Ae. aegypti, a single haplotype was observed for ITS2.BLAST analysis of ITS2 revealed 100% identity match for multiple species within Aedes genus.Phylogenetic analysis of the ITS2 gene confirmed the genus of the specimen as Aedes but did not provide strong bootstrap support for species level identification.For COI, nineteen different haplotypes were identified, sixteen among the Kebridehar samples and five among the Degehabur samples.The COI tree confirmed the species as Ae.aegypti (

Discussion
Aedes aegypti is considered the main vector of dengue viruses and other arboviral diseases worldwide.Therefore, entomological study was carried out to identify the presence of Ae. aegypti in urban areas of the Somali region and to study its seasonality and breeding habitats in Kebridehar town.Our data provides a baseline for tracking the transmission of arboviruses in the Somali region.Our study revealed that Ae. aegypti was documented in all study sites   such as Jigjiga, Degehabur, Kebridehar and Godey, it is widely spread across sub-Saharan Africa and Asian countries [15,16].Similarly, in Ethiopia, this species was detected in urban areas in Dire Dawa [10,17], Metema, Humera [18] and Kebridehar [13].The presence of this mosquito in each month surveyed indicates an established Aedes mosquito population in Kebridehar.Recent outbreaks of dengue and chikungunya have been reported in Kebridehar and Dire Dawa [13].
Analysis of the mitochondrial COI gene provided a good confirmation of species identification and revealed multiple COI haplotypes exists among the Ethiopian Ae. aegypti.More genomic data is needed for deeper phylogeographic analysis.While some support for subpopulation differentiation was observed among some Degehabur samples (bootstrap = 84), most Ae.aegypti in Ethiopia and database sequences representing Kenya, Australia, and Indonesia clustered into a large branch with significant support for distinction from Ae. albopictus.The high level of variation supports the evidence, Ae. aegypti abundance in east Ethiopia of a long-established population.
In the phylogenetic analysis, the set of samples from Ethiopia grouped away from the Australian and Indonesian samples with a significant bootstrap value of 73.On the other hand, the Kenyan samples grouped with the Ethiopian samples suggesting a close relationship due to the geographic proximity.Overall, this phylogenetic tree indicates the origin of the Aedes population in Ethiopia is more likely to be from a neighboring country.The differentiation trends (no significant bootstrap values) among Ethiopian samples suggest the absence of subpopulations of Ae. aegypti among our collection in Ethiopia, though further sequencing is needed to be definitive.To further investigate the origin of the Ethiopian Aedes populations studied here, we need to explore the level of differentiation between subpopulations within Ethiopia, and we need to compare the level of differentiation within Ethiopia to the differentiation between Ethiopia and neighboring countries.
An essential component of vector competence is an understanding of the magnitude of temporal variability and consistency.We assessed the seasonality of Ae. aegypti for the first time in Somali region of Ethiopia.A high number of Ae. aegypti were detected during the wet season, with highest number detected in November, followed by October, and December.Similar studies in West Africa found that adult Ae. aegypti population numbers often show a positive correlation with rainfall with a single peak in abundance in areas with a single wet season [19,20].A high density of Aedes mosquito species was observed during the wet season compared to the dry season in Cameroon [21].However, a study in Ghana showed that Aedes populations increased during the dry season rather than the wet season [22].In our study, the detection during Ethiopia's dry season may be due to the presence of water storage containers in every house serving as breeding habitats.
The present study showed that Ae. aegypti larvae and pupae were found in discarded tires, cemented cisterns, plastic containers, and barrels.All the positive water holding containers for Aedes mosquito were found outside houses which was 10m radius.In addition, the Larval Indices found in this survey were relatively high.These values were similar to what has been reported in Dire Dawa, Metema and Humera [10,18].High Larval Indices were also reported in Kebridehar in 2017 that coincided with an outbreak of dengue fever [13].The high Larval Indices in Kebridehar in this study suggests a persistent high risk for dengue outbreaks.Good environmental sanitation and a consistent water supply system would reduce the number of putative Ae. aegypti breeding sites [23] particular in Kebridehar which had the highest larval indices.
In our study, discarded tires contained the most Aedes mosquitoes.These discarded tires that were distributed throughout Kebridehar Town were found placed outdoors, stationary for an extended period of time, and filled with water during the rainy season.These factors combined made for ideal mosquito breeding habitats.Our finding highlight discarded tires as a major breeding source is consistent with previous studies conducted in Dire Dawa, Metema and Humera, Ethiopia [10,17,18] and in Tanzania, Zanzibar, and Mozambique [23,24,25].Collectively, these findings point to the threat of discarded tires and more interventions involving the appropriate handling of old tires should be implemented.
Our study also revealed that Aedes were also breeding in cemented cistern water containers.Outdoor cemented cistern water containers (local named as Birka) are present with most houses in Kebridehar Town as water reservoirs in the absence of an efficient water system.This water is brought from a long distance due to the scarcity of water around the town which is a dry area, and people used this water for drinking and daily consumption.The role of these types of larger water containers for Aedes breeding and a driver to the movement of Aedes was previously reported (i.e., birka) in Dire Dawa city [17].We observed that majority of cemented cistern water containers those build for drinking purposes were well covered with corrugated iron but the stored water is being kept for more than a month and this favors Aedes and other mosquito spp. to breed and spread throughout the town which likely could attribute for high diseases transmission and the possibility to increase outbreaks.Following that, evidence revealed that frequent outbreaks of dengue and chikungunya were investigated in Kebridehar town due to an increase in vectors in the town [13].More studies are needed to determine how impactful these cisterns are for the spread of Ae. aegypti in Ethiopia.
Another potential source of Aedes larvae and pupae in Kebridehar Town was plastic containers.It is well known that Kebridehar Town is urban and growing and expanding, many house constructions were underway.Many houses were being built, and water was being stored in plastic containers for construction.Because these containers were open, exposed, and static for a long time with poor handling, it is likely that mosquitoes used them to lay their eggs.If larvae and pupae were able to survive and develop into adult mosquitoes, there would be significant disease transmission in the town.In similar studies, Ae. aegypti was more prevalent in plastic containers used for storing water in Cameron [21].Therefore, the local government and the regional health bureau must act when there is construction in the town.People and contractors should be aware that when they build plastic containers and other water storage, the water containers need to be covered, kept clean, and replaced right away.
Interestingly, we also identified two species in the Somali Region not previously investigated including Cs. longiarelota and Ae.hirsutus.These two species were found in plastic water containers built for construction purposes similarly to what was reported previously [26].Cs. longiarelota species has been reported in the Middle East [27] and is invasive to Europe [26].Cs. longiarelota is reported vector of West Nile virus.While Ae. hirsutus is not a known as a vector of human diseases, further investigation of this species in needed.Our findings highlight the importance of incorporating molecular identification into Aedes surveillance to identify other potential vectors of harmful diseases.

Conclusion
The study provides preliminary evidence of Ae. aegypti in all study sites including Jigjiga, Degehabur, Kebridehar and Godey towns.The results show that Ae. aegypti were found close to human due to the presence of breeding habitat like cemented water reservoir (local name called Birka), tires, plastic containers, and barrels inside human dwelling.The high observed values of the Aedes mosquito larval indices suggest a high risk of arbovirus transmission when arboviral cases become established in the area.Therefore, early interventions are necessary to combat the burden of emerging arboviral diseases and it is critical planning and implementation of vector control strategy in the region.Further, Ae. aegypti control programs should concentrate their interventions on the education and engagement of residents in appropriate use and disposal of old tires and covering of water holding containers.
Fig 6) with strong bootstrap support (bs = 98).The unknown specimens collected in Jigjiga in 2022 (S1 and S2 Figs) were genetically analyzed separately.BLAST analysis of COI returned Culiseta longiareolata and Aedes hirsutus.Phylogenetic analysis of the COI sequences for each species along with database sequences

Table 2 . Larval density, adults emerged from larvae and breeding site indices for each breeding site container type in Kebridehar. Cisterns Tires Plastic containers Barrels Water pool Overall total* or average**
* ǂ RBI relative breeding index (number of habitats positive for Ae.aegypti divided by the total number of habitats positive for any mosquito) https://doi.org/10.1371/journal.pone.0296406.t002